Recombinant bacterial lipocalin blc and uses thereof

ABSTRACT

The present inventors have solved the crystal structure of an  Escherichia coli  bacterial lipocalin polypeptide, which depicts a monomeric protein. Previous crystal structures have been reported, but these appear to be inaccurate, as they predicted, e.g., a dimeric protein. The crystal structure of a bacterial lipocalin provided by the present invention leads to numerous uses. For example, the present invention provides for the design, construction and use of recombinant libraries of diversified bacterial lipocalins resulting from a bacterial lipocalin polypeptide “backbone”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/EP2011/065423,filed Sep. 7, 2011, which claims priority from U.S. ProvisionalApplication No. 61/380,521, filed Sep. 7, 2010.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 4, 2013, isnamed sequence.txt and is 10 KB.

BACKGROUND

Lipocalins represent a family of functionally diverse, small proteinscomprising 160-180 residues that share high conservation at the tertiarystructural level while having weak amino acid sequence homology (Floweret al., 2000; Skerra, 2000). Their fold is dominated by aneight-stranded antiparallel β-barrel with an α-helix attached to itsside, whereby four structurally variable loops, which connectneighboring β-strands at the open end of the barrel, form the entranceto a ligand pocket. Further hallmarks are three structurally conservedregions (SCRs) (Flower, 1996), which assist the identification of newlipocalins at the primary structure level.

Lipocalins were initially described for eukaryotes and only morerecently identified in Gram-negative bacteria (Flower, 1996; Bishop,2000). The bacterial lipocalin (Blc) was first discovered in Escherichiacoli (Bishop et al., 1995), but sequence analyses have indicated theexistence of at least 20 other bacterial lipocalins, for example inCitrobacter freundii, Vibrio cholerae and many other Enterobacteriaceae.

Blc belongs to the class I outer membrane lipoproteins, carrying a typeII signal peptide at the N-terminus, which directs export into theperiplasm. After signal peptide processing, the protein becomes anchoredinto the inner leaflet of the outer membrane (Bishop et al., 1995) via alipid-modified amino-terminal cysteine residue. The blc promoter ismainly induced at the onset of the stationary growth phase via the rpoSsigma factor, which generally directs gene expression for adaptation tostarvation and high osmolarity or other conditions known to exert stresson the cell envelope. The blc gene is poorly transcribed, suggestingthat the normal concentration of Blc in the outer membrane is low.

Other findings indicate an implication of Blc in bacterial hostpathogenesis (Bishop, 2000). The blc genes of some Enterobacteriaceaeare physically linked to the ampC gene, which encodes a serineβ-lactamase on the chromosome but also appears to be geneticallyrecombined into different plasmids. This co-localisation suggests thatthe blc gene may be involved in antibiotic resistance. Furthermore,bacterial lipocalins play a role in the host immune response as manycomponents of the bacterial cell envelope provide so-calledpathogen-associated molecular patterns for surveillance. One relevantcomponent is the N-acyl-S-sn-1,2-diacylglycerylcysteine modification atthe N-terminus of the bacterial lipoproteins, permitting macrophages andother immune cells to recognize Blc via CD14 and the Toll-like receptor2.

The first crystal structure of an N-terminally extended version of aBlc, so-called Blc-X (Campanacci et al. 2004), revealed the β-barrelfold characteristic for the lipocalin family and was followed by asecond crystal structure of Blc-X in complex with the fatty acidvaccenic acid (Campanacci et al., 2006). Both structures belong to thespace group P2₁2₁2₁ with isomorphous unit cell parameters and an overallr.m.s. deviation of 0.1 Å for 167 Cα atoms.

The crystal structures of Campanacci et al. indicated that Blc was adimeric protein. This prediction was based on the identification of atight pairwise contact of several side chains and a buried surface of786 Å² and 825 Å², respectively, of the two distinct Blc-X molecules Aand B within the asymmetric unit, and also on static light scatteringmeasurements in solution. Notably, the fatty acid ligand was bound inthe cavity of just one molecule of the dimer and involved in only a fewadditional contacts to the other molecule, which was explained by theasymmetric interaction of the two Blc-X monomers. Yet, binding ofvaccenic acid did not lead to detectable conformational changes withinthe Blc-X dimer (Campanacci et al., 2006).

The present inventors, however, studied the biochemistry and structureof a recombinant Blc without N-terminal extension overproduced with adifferent E. coli expression vector, and found striking evidence thatBlc behaves as a stable monomer in solution. The previously describeddimerization is, therefore, likely the result of a cloning artifact.This surprising finding is one of the bases of the present invention.

SUMMARY OF THE INVENTION

The present invention can be summarized by the following items:

-   1. A method of preparing a plurality of nucleic acid molecules,    comprising the step of (i) synthesizing or (ii) recombinantly    producing a plurality of nucleic acid molecules, wherein said    molecules differ from each other in at least one nucleotide at a    position within at least one loop region of the nucleic acid    encoding a Blc polypeptide.-   2. A method according to item 1, comprising the step of synthesizing    or recombinantly producing at least six different nucleic acid    molecules,-   3. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within at least two loop regions of the nucleic acid    encoding said Blc polypeptide.-   4. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within at least three loop regions of the nucleic acid    encoding said Blc polypeptide.-   5. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within at least four loop regions of the nucleic acid    encoding said Blc polypeptide.-   6. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within the nucleotides encoding amino acid residue    positions 88-96 of said Blc polypeptide, and wherein said molecules    encode different polypeptides from each other.-   7. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within the nucleotides encoding amino acid residue    positions 27-43 of said Blc polypeptide, and wherein said molecules    encode different polypeptides from each other.-   8. A method according to any one of the preceding items, wherein    said molecules differ from each other in at least one nucleotide at    a position within the nucleotides encoding amino acid residue    positions 58-73 of said Blc polypeptide, and wherein said molecules    encode different polypeptides from each other.-   9. A method according to any one of any one of the preceding items,    wherein said molecules differ from each other in at least one    nucleotide at a position within the nucleotides encoding amino acid    residue positions 113-121 of said Blc polypeptide and wherein said    molecules encode different polypeptides from each other.-   10. A method of preparing a plurality of polypeptide molecules,    comprising the steps of expressing the plurality of nucleic acid    molecules according to any one of the preceding items.-   11. A composition comprising a plurality of nucleic acid molecules,    wherein said molecules differ from each other in at least one    nucleic acid nucleotide at a position within at least one loop    region of the nucleic acid encoding a Blc polypeptide.-   12. A composition according to item 11, wherein said plurality    comprises at least six different nucleic acid molecules.-   13. A composition according to item 11 or 12, wherein said nucleic    acid molecules differ from each other in at least one nucleotide at    a position within at least two loop regions of the nucleic acid    encoding said Blc polypeptide.-   14. A composition according to any one of items 11 to 13, wherein    said molecules differ from each other in at least one position    within at least three loop regions of the nucleic acid encoding said    Blc polypeptide.-   15. A composition according to any one of items 11 to 14, wherein    said molecules differ from each other in at least one nucleotide at    a position within at least four loop regions of the nucleic acid    encoding said Blc polypeptide.-   16. A composition according to any one of items 11 to 15, wherein    said molecules differ from each other in at least one nucleotide at    a position within the nucleotides encoding amino acid residue    positions 88-96 of said Blc polypeptide, and wherein said molecules    encode different polypeptides from each other.-   17. A composition according to any one of items 11 to 16, wherein    said molecules differ from each other in at least one nucleotide at    a position within the nucleotides encoding amino acid residue    positions 27-43 of said Blc polypeptide.-   18. A method according to any one of items 11 to 17, wherein said    molecules differ from each other in at least one nucleotide at a    position within the nucleotides encoding amino acid residue    positions 58-73 of said Blc polypeptide-   19. A method according to any one of items 11 to 18, wherein said    molecules differ from each other in at least one nucleotide at a    position within the nucleotides encoding amino acid residue    positions 113-121 of said Blc polypeptide.-   20. A composition comprising a plurality of polypeptide molecules,    wherein said polypeptide molecules differ from each other in at    least one amino acid at a position within at least one loop region    of a Blc polypeptide.-   21. A composition according to item 20, wherein said plurality    comprises at least six different polypeptide molecules.-   22. A composition according to item 20 or 21, wherein said    polypeptide molecules differ from each other in at least one amino    acid at a position within at least two loop regions of said Blc    polypeptide.-   23. A composition according to any one of items 20 to 22, wherein    said polypeptide molecules differ from each other in at least one    amino acid at a position within at least three loop regions of said    Blc polypeptide.-   24. A composition according to any one of items 20 to 23, wherein    said polypeptide molecules differ from each other in at least one    amino acid at a position within at least four loop regions of said    Blc polypeptide-   25. A composition according to any one of items 20 to 24, wherein    said molecules differ from each other in at least one amino acid    position within amino acid residue positions 88-96 of said Blc    polypeptide.-   26. A composition according to any one of items 20 to 25, wherein    said molecules differ from each other in at least one amino acid    position within amino acid residue positions 27-43 of said Blc    polypeptide.-   27. A method according to any one of items 20 to 26, wherein said    molecules differ from each other in at least one amino acid position    within amino acid residue positions 58-73 of said Blc polypeptide-   28. A method according to any one of items 20 to 27, wherein said    molecules differ from each other in at least one amino acid position    within amino acid residue positions 113-121 of said Blc polypeptide.-   29. A method of isolating a polypeptide of interest, comprising the    steps of (i) allowing the composition of any one of items 20 to 28    to be in contact with a target of interest, wherein said polypeptide    of interest is comprised within said composition and specifically    binds to the target; and (ii) isolating the polypeptide or a    polypeptide-target complex resulting from said specific binding.-   30. A method of isolating a nucleic acid molecule of interest,    comprising the steps of (i) allowing the composition of any one of    items 20 to 28 to be in contact with a target of interest, where    said polypeptide is comprised within said composition and    specifically binds to the target; (ii) isolating the polypeptide or    a polypeptide-target complex resulting from said specific    binding; (iii) isolating the nucleic acid sequence encoding said    polypeptide; and (iv) determining the nucleic acid encoding said    polypeptide.-   31. A crystal structure of a monomeric Blc polypeptide.-   32. A crystal structure according to item 31, wherein said structure    is depicted in FIG. 3.-   33. A method according to any one of items 1-10, 18-17, and 27-30    wherein said molecules are selected from a group consisting of at    least 50%, at least 60%, at least 75%, at least 90%, at least 95%,    or at least 99% sequence identity with SEQ-ID NO. 1,-   34. A composition according to any one of items 11-17 and 20-26    wherein said molecules are selected from a group consisting of at    least 50%, at least 60%, at least 75%, at least 90%, at least 95%,    or at least 99% sequence identity with SEQ-ID NO. 1.-   35. An isolated crystalline form of a monomeric Blc polypeptide-   36. An isolated crystalline form of item 35, wherein said    crystalline form has a I4122 space group and unit cell dimensions    a=b=88.9, c=78.4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of Blc (dark grey) crystallized in space group I4₁22,with Blc-X (light grey) crystallized in space group P2₁2₁2₁ (PDB entry2ACO, depicting the two monomers in the asymmetric unit) aftersuperposition of the 149 Cα positions resolved for all structures. Thepreviously published structure of Blc-X reveals a long N-terminalextension including an additional β-strand and a hairpin loop (black).N-Blc denotes the first residue visible in the crystal structure forBlc, N-BlcX denotes the first residue visible in the crystal structurefor Blc-X, C labels the C-termini of all three structures shown.

FIG. 2: Analysis of the dimer interface in the previously describedBlc-X crystal structure with two monomers in the asymmetric unit (PDBentry 2ACO). The ligand vaccenic acid (labeled VCA, grey spheres)occupies only one ligand pocket. The N-terminal peptide extension ofBlc-X, which includes the hairpin loop and the additional β-strandoutside the β-barrel, is colored black for both monomers. The fourmutated residues within the hairpin loop of Blc-X (Lys-Ala-Gly-Ser),whose sequence positions correspond to the four N-terminal amino acidsof mature wild-type Blc, are depicted with side chains and labeled forthe rightmost monomer B. This segment of molecule B forms directcontacts with three side chains (Tyr94, Lys105, Tyr113) displayed on theβ-barrel surface of molecule A (left). Next to this interaction, theE/F-loops of both molecules form another tight, almost symmetricalcontact. N- and C-termini of the polypeptide chains are labeled.

FIG. 3: Crystalline structure of the invention elucidated by X-raydiffraction. Amino acid positions 27-43, 58-73, 88-96, 113-121 representthe loop regions of the mature E. coli Blc.

FIG. 4: SEQ ID NO. 1: Full length recombinant Blc as encoded on pBlc2,including the OmpA signal peptide (amino acid residues 1-21) and theC-terminal Strep-tag.

DETAILED DESCRIPTION

Unless otherwise specified, “a” or “an” means “one or more.” Thedisclosure illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Also, the term “comprising” when used herein canbe replaced by the term “consisting of”. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible. Thus, it should be understood that althoughthe present disclosure has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of thedisclosures embodied therein herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this disclosure. The compositions,methods, procedures, treatments, molecules and specific compoundsdescribed herein are presently representative of preferred embodimentsare exemplary and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention aredefined by the scope of the claims. The listing or discussion of apreviously published document in this specification should notnecessarily be taken as an acknowledgement that the document is part ofthe state of the art or is common general knowledge.

The present inventors have solved the crystal structure of a particularEscherichia coli bacterial lipocalin (Blc), as depicted in dark grey inFIG. 1, which allows for several novel application as further describedherein. As used herein, “Blc” is defined as any bacterial lipocalin,including but not limited to the bacterial lipocalin having SwissProtEntry P0A901. Blc polypeptides comprise a class of polypeptides thatbelongs to the class I outer membrane lipoproteins, carrying a type IIsignal peptide at the N-terminus which allows export into the periplasm.The amino acid sequence of the Blc polypeptide is shown in SEQ ID NO. 3,with amino acids 1-18 representing the type II signal peptide and aminoacids 19-177 representing the mature bacterial lipocalin protein. Aftersignal peptide processing, the protein becomes anchored into the innerleaflet of the outer membrane (Bishop et al., 1995) via a lipid-modifiedamino-terminal cysteine residue. SEQ ID NO. 4 shows the amino acidsequence of the mature bacterial lipocalin protein.

Accordingly, the invention relates to a method of preparing a pluralityof nucleic acid molecules, comprising the step of (i) synthesizing or(ii) recombinantly producing a plurality of nucleic acid molecules,wherein said molecules differ from each other in at least one nucleotideat a position within at least one loop region of the nucleic acidencoding a Blc polypeptide. The nucleic acids of the plurality ofnucleic acids encode one or more bacterial lipocalin (Blc) muteins.

Also, the invention relates to a composition comprising a plurality ofnucleic acid molecules, wherein said molecules differ from each other inat least one nucleic acid nucleotide at a position within at least oneloop region of the nucleic acid encoding a Blc polypeptide. Similarly,the invention relates to a plurality of polypeptide molecules, whereinsaid polypeptide molecules differ from each other in at least one aminoacid at a position within at least one loop region of a Blc polypeptide.These polypeptide molecules are in the sense of the invention bacteriallipocalin muteins.

Additional embodiments relate to novel crystals of monomer Blc.

The amino acid sequence shown in SEQ ID NO. 1 (or 2) comprises at aminoacid position 22-180 the mature bacterial lipocalin protein, while aminoacids 1-21 represent the type I OmpA signal peptide and amino acids181-189 represent the Strep-tag II.

For the purpose of the present invention whenever reference is made tobacterial lipocalin, the mature bacterial lipocalin shown, for example,in SEQ ID NO. 1 at amino acid positions 22-180), SEQ ID NO. 2 at aminoacid positions 22-180, SEQ ID NO. 3 at amino acid positions 19-177 orSEQ ID NO. 4 at amino acid positions 1-159 is meant. Thus, whenlocating, for example, the amino acid residue positions 27-43, 58-73,88-96 or 113-121 of the loops of the bacterial lipocalin of the presentinvention should be located, the starting point for counting is in SEQID NO. 1 and SEQ ID NO. 2 amino acid position 22 (i.e., position 22 ofSEQ ID NO. 1 or 2 is position “1” for that purpose), in SEQ ID NO. 3amino acid position 19 (i.e., position 19 of SEQ ID NO. 3 is position“1” for that purpose) and in SEQ ID NO. 4 amino acid position 1 (i.e.,position 1 of SEQ ID NO. 4 is position “1” for that purpose).

In one aspect, the present invention relates to a library of functionalpolypeptides that is based on the Blc lipocalin; accordingly, theinvention includes polypeptides that have at least 50%, preferably atleast 75%, more preferably at least 90%, and most preferably at least99% sequence identity with SEQ.ID NO. 1 or 3. It is preferred that thedegree of identity is determined over the full length of the sequencebeing compared. It is also preferred that polypeptides that have thedegree identity as described herein with SEQ ID NO. 1 or 3 have one ormore amino acid replacements, in particular in the loops (1, 2, 3 and/or4) in comparison to the wildtype bacterial lipocalin. The use of variouslipocalins as a “progenitor” to develop a library of lipocalinpolypeptides that have diversified amino acids compared to theprogenitor has been described (see, e.g., Beste et al, (1999)). Indeed,libraries of human lipocalin-derived proteins, called Anticalins, haveled to the isolation of several therapeutically relevant drug candidates(see, e.g. WO05/019256, WO06/056464).

Prior to the proper elucidation of the Blc polypeptide by the presentinventors, however, a rational design of a diverse library ofBlc-derived Anticalins was not possible. For instance, based on thepreviously proposed crystal structure of Blc, Campanacci et al.predicted that Blc would former homodimers, which would not havemotivated the skilled worker to design a library based on Blc, given,e.g., the inherent complexities of utilizing a dimeric lipocalin overthe known monomeric lipocalins whose structures already have beensolved.

According to the previously reported structures, as shown in FIG. 2, theskilled worker would have been motivated, for example, againstdiversifying the E/F and G/H loop regions in a library of diversifiedBlc polypeptides, as the skilled worker would have expected theseregions to form part of, or be close to, the interface of the proposedBlc dimer.

Accordingly, an accurate crystal structure of a bacterial lipocalin,which is one aspect of the present invention, provides numerousapplications, which also are provided by the present invention. Forexample, the present invention provides for the design, construction anduse of recombinant libraries of diversified bacterial lipocalins(including nucleic acid molecules encoding the same) modelled after abacterial lipocalin polypeptide “backbone” such as Blc. Techniques forrecombinantly producing proteins are known to those skilled in the art(e.g. Skerra, A. (2001)).

Thus, in general, the present invention contemplates a monomeric Blcprotein having the amino acid sequence shown in SEQ ID NO. 3. Also, itis generally preferred that the bacterial lipocalin muteins of thepresent invention are monomeric.

In one embodiment, the present invention provides a method of preparinga plurality of nucleic acid molecules that are based on a Blcpolypeptide and that differ from each other.

As used herein, a “plurality” is defined as two or more. In a preferredembodiment, the plurality includes at least six nucleic molecules andmay include at least 50, 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9,10^10, or at least 10^11 nucleic acid molecules.

The present invention also contemplates a method of preparing aplurality of polypeptide molecules that are based on a Blc polypeptideand that differ from each other. In this sense, the present inventionincludes a method of expressing the plurality of nucleic acids moleculesthat are based on a Blc polypeptide and contemplated herein.

Additionally, the present invention provides compositions that include aplurality of nucleic acid molecules that are based on a Blc polypeptideand that differ from each other. Within the plurality of nucleic acidmolecules contemplated by the present invention (as well as the methodsdescribed herein), the molecules may differ from each other in anyregion. The plurality of nucleic acid molecules may, for example, differin a loop region of a Blc polypeptide. In this sense, the plurality ofnucleic acid molecules may, for instance, differ in at least onenucleotide at a position within one, two, three and/or four loop regionsof the nucleic acid encoding a Blc polypeptide. These positions may, butare not required to, include at least one nucleotide at a positionwithin the nucleotides encoding amino acid residue positions 27-43,58-73, 88-96, 113-121 of a Blc polypeptide. Preferably, said molecules,substantially all of said molecules or the majority of said moleculesencodes different polypeptides from each other. As used herein, a “loopregion” of a Blc polypeptide is defined as a peptide segment which joinstwo adjacent β-strands that form part of the β-barrel in the lipocalinstructure, which may include adjoining parts of the β-strandsthemselves. Examples for such loop regions are the segments comprisingamino acid residue positions 27-43, 58-73, 88-96 or 113-121 of a Blcpolypeptide.

The amino acid sequence for wild type Blc, including its signalsequence, (SEQ ID NO:3) is:

  1 mrllplvaaa taaflvvaCS SPTPPRGVTV VNNFDAKRYL GTWYEIARFD HRFERGLEKV 61 TATYSLRDDG GLNVINKGYN PDRGMWQQSE GKAYFTGAPT RAALKVSFFG PFYGGYNVIA121 LDREYRHALV CGPDRDYLWI LSRTPTISDE VKQEMLAVAT REGFDVSKFI WVQQPGS

The lower-case letters represent the signal peptide, while theunderlined letters represent the Blc loops that can be randomizedaccording to the present invention.

Additionally, the present invention provides compositions that include aplurality of polypeptides. In this sense, the present inventionprovides, for example, compositions that include a plurality of aminoacid sequences that are based on a Blc polypeptide and that differ fromeach other. Within the plurality of polypeptides and, hence, amino acidsequences contemplated by the present invention (as well as the methodsdescribed herein), the molecules may differ from each other in anyregion. The plurality of polypeptides may, for example, differ in a loopregion of a Blc polypeptide. In this sense, the plurality ofpolypeptides may, for instance, differ in at least one amino acid at aposition within one, two, three and/or four loop regions of a Blcpolypeptide. These positions may, but are not required to, include atleast one position within the amino acid residue positions 27-43, 58-73,88-96, 113-121 of a Blc polypeptide. Preferably, said molecules,substantially all of said molecules or the majority of said moleculesencodes different polypeptides from each other.

The present invention also contemplates the preparation of a library ofBlc variants that contains a plurality of polypeptide molecules, wherepolypeptide molecules may contain one or more different amino acids(vis-a-vis a mature Blc polypeptide) within any or all of the 4 loopregion positions, as delineated in FIG. 3.

The present invention also provides a method of isolating a polypeptideof interest, as described in Kim et al. (2009) and Schönfeld et al.(2009). In one aspect, this includes a step of allowing a composition ofthe present invention to be in contact with a target of interest. Tothis end, a polypeptide of interest is comprised within the compositionand specifically binds to the target. As used herein, a “target” isdefined as any molecule to which a polypeptide of the invention iscapable of specifically binding, including all types of proteinaceousand non-proteinacious molecules such as haptens or other smallmolecules. As used herein, a polypeptide of the invention “specificallybinds” a target if it is able to discriminate between that target andone or more reference targets, since binding specificity is not anabsolute, but a relative property. “Specific binding” can be determined,for example, in accordance with Western blots, ELISA-, RIA-, ECL-,IRMA-tests, FACS, IHC and peptide scans. The polypeptide of theinvention can bind to the target with an affinity in the micromolar or,in more preferred embodiments, in the nanomolar range. Binding constantsof less than 100 μM, 50 μM, 500 nM, 250 nM, 100 nM and 50 nM are alsoenvisioned for the current invention.

According to this method, a polypeptide that specifically binds to atarget of interest is preferably isolated, which can be accomplishedaccording to conventional techniques, such as those defined in Kim etal. (2009) and Schönfeld et al. (2009), which typically involve theselection of a polypeptide of interest (phenotype) that is linked to itsunderlying genetic code (genotype). For instance, the polypeptide ofinterest may be expressed and then selected as part of any conventionalselection technology, for example, a technology that involves areplicable genetic package, such as a bacteriophage. These technologiesinclude, for example, display technologies such as phage display andcell surface display. In vitro selection technologies, such as ribosomedisplay, also may be used. See, e.g., Kawasaki, U.S. Pat. Nos. 5,643,768and 5,658,754.

The present invention also provides a crystal structure of a monomericBlc polypeptide. In a particular embodiment, the present inventionincludes the crystal structure depicted in FIGS. 1 and 3. The presentinvention also includes a Blc crystal being characterized by the datashown in Table 1.

A preferred embodiment of the method for preparing a plurality ofnucleic acid molecules includes the generation of a mutein of abacterial lipocalin protein, said mutein having detectable affinity to agiven target, comprising the step of (a) subjecting the bacteriallipocalin to mutagenesis at one or more of the sequence positions whichcorrespond to the sequence positions 27 to 43 (loop 1), 58-73 (loop 2),88-96 (loop 3) and/or 113 to 121 (loop 4) of SEQ ID NO. 3, resulting inone or more mutein (s) of the bacterial lipocalin protein.

In a preferred embodiment, said method further comprises step (b)enriching at least one resulting mutein having binding affinity for agiven target from the one or more muteins by selection and/or isolatingsaid at least one mutein.

Preferably, the mutagenesis in step (a) of the method for generating amutein of a bacterial lipocalin results in a plurality of muteins of theprotein.

It is also a preferred embodiment of the present invention that furtheramino acids of the bacterial lipocalin protein (apart from thosecomprised by the four loops) are subjected to mutagenesis in the abovemethod.

The composition of the present invention comprising a plurality ofpolypeptide molecules preferably includes a Blc mutein in which one ormore amino acids within one, two, three, or all four loops are changedin comparison to the wildtype (or reference) Blc of the presentinvention. Said one or more amino acids include 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids thatcan be changed in loop 1, loop 2, loop 3 and loop 4. However, it is alsoenvisaged that within loop1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, or 17; within loop2 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16; within loop3 1, 2, 3, 4, 5, 6, 7, 8, or 9; and/orwithin loop4 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are replaced.“Replacement” means that an amino acid different from that present atthe corresponding position in the wildtype (or reference) bacteriallipocalin is present in a bacterial lipocalin of the present invention.

The present invention also envisages a bacterial lipocalin other thanBlc in which one or more amino acids within one, two, three, or all fourloops are changed in comparison to the wildtype (or reference) bacteriallipocalin of the present invention. Said one or more amino acids include1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore amino acids that can be changed in loop 1, loop 2, loop3 and loop4. However, it is also envisaged that within loop1 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; within loop2 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; within loop3 1, 2, 3, 4, 5, 6,7, 8, or 9; and/or within loop4 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acidsare replaced.

The loops of a bacterial lipocalin other than Blc can be readilyidentified by the skilled person as described herein. Briefly, theskilled person can align the Blc of the present invention and an aminoacid sequence of interest that is assumed to be a bacterial lipocalin soas to determine (i) the degree of identity and/or (ii) the position ofthe loops that correspond to those of the loops of Blc.

When used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid) or “mutant” refers to the exchange, deletion, or insertionof one or more nucleotides or amino acids, respectively, within thebacterial lipocalin protein (Blc) of the present invention compared tothe naturally occurring (wild-type) nucleic acid or protein “reference”scaffold of Blc, for example, shown in SEQ ID NO. 3.

Accordingly, a mutein of the invention may include the wild type(natural) amino acid sequence of the “parental” protein scaffold(bacterial lipocalin (Blc)) outside the mutated one or more amino acidsequence positions within one, two, three or four loop(s);alternatively, a bacterial lipocalin mutein may also contain amino acidmutations outside the sequence positions subjected to mutagenesis thatdo not interfere with the binding activity and the folding of themutein. Such mutations can be accomplished on a DNA level usingestablished standard methods (Sambrook, J. et al. (2001) MolecularCloning: A Laboratory Manual, 3^(rd) Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Possible alterations of the amino acidsequence are insertions or deletions as well as amino acidsubstitutions.

Such substitutions may be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. One the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of a parental protein scaffold, where thesedeletions or insertion result in a stable folded/functional mutein,which can be readily tested by the skilled worker.

The skilled worker will appreciate methods useful to prepare proteinmuteins contemplated by the present invention but whose protein ornucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence include, e.g.,directed mutagenesis of single amino acid positions in order to simplifysub-cloning of a mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for a given target. Furthermore, mutations can beintroduced to modulate certain characteristics of the mutein such as toimprove folding stability, serum stability, protein resistance or watersolubility or to reduce aggregation tendency, if necessary. For example,naturally occurring cysteine residues may be mutated to other aminoacids to prevent disulphide bridge formation.

Accordingly, the invention also includes functional variants of muteinsdisclosed herein, which have a threshold sequence identity or sequencehomology to a reference protein. By “identity” or “sequence identity” ismeant a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent invention means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the invention with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Percent identity isdetermined by dividing the number of identical residues by the totalnumber of residues and multiplying the product by 100. The term“homology” is used herein in its usual meaning and includes identicalamino acids as well as amino acids which are regarded to be conservativesubstitutions (for example, exchange of a glutamate residue by anaspartate residue) at equivalent positions in the linear amino acidsequence of two proteins. Most preferred, the amino acid sequence shownin SEQ ID NO. 3 is preferred as a “reference sequence”. SEQ ID NO. 3shows the mature bacterial lipocalin protein (Blc). The term “referencesequence” and “wild type sequence” (of Blc) is used interchangeablyherein.

As mentioned herein, the present invention relates to polypeptides thatshare a certain degree of identity with the bacterial lipocalin protein(Blc) of the present invention. Such polypeptides comprise preferablyone or more amino acid replacements in comparison to the wildtype Blcamino acid sequence.

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1; cutoff value set to 10⁻³) including the propeptidesequences, preferably using the wild type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

It is also possible to deliberately mutate other amino acid sequencepositions to cysteine in order to introduce new reactive groups, forexample, for the conjugation to other compounds, such as polyethyleneglycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins,or for the formation of non-naturally occurring disulphide linkages.With respect to a mutein of human Lipocalin 2, exemplary possibilitiesof such a mutation to introduce a cysteine residue into the amino acidsequence of a bacterial lipocalin mutein.

The term “position” when used in accordance with the invention means theposition of either an amino acid within an amino acid sequence depictedherein or the position of a nucleotide within a nucleic acid sequencedepicted herein. The term “corresponding” as used herein also includesthat a position is not only determined by the number of the precedingnucleotides/amino acids. Accordingly, the position of a given amino acidin accordance with the invention which may be substituted may vary dueto deletion or addition of amino acids elsewhere in a (mutant orwild-type) lipocalin. Similarly, the position of a given nucleotide inaccordance with the present invention which may be substituted may varydue to deletions or additional nucleotides elsewhere in a mutein or wildtype bacterial lipocalin 5′-untranslated region (UTR) including thepromoter and/or any other regulatory sequences or gene (including exonsand introns).

Thus, under a “corresponding position” in accordance with the inventionit is preferably to be understood that nucleotides/amino acids maydiffer in the indicated number but may still have similar neighboringnucleotides/amino acids. Said nucleotides/amino acids which may beexchanged, deleted or added are also comprised by the term“corresponding position”. When used herein “at a position correspondingto a position” a position in a “query” amino acid (or nucleotide)sequence is meant that corresponds to a position in a “subject” aminoacid (or nucleotide) sequence.

Specifically, in order to determine whether a nucleotide residue oramino acid residue of the amino acid sequence of a bacterial lipocalindifferent from a bacterial lipocalin mutein of the invention correspondsto a certain position in the nucleotide sequence or the amino acidsequence of a bacterial lipocalin mutein as described, in particular anyof SEQ ID NOs. 1-4, a skilled artisan can use means and methodswell-known in the art, e.g., alignments, either manually or by usingcomputer programs such as BLAST 2.0 (Altschul et al. (1990), J. Mol.Biol. 215:403-10), which stands for Basic Local Alignment Search Tool,or ClustalW (Thompson et al. (1994), Nucleic Acids Res. 22(22):4673-80)or any other suitable program which is suitable to generate sequencealignments. Accordingly, a bacterial lipocalin mutein of any of SEQ IDNOs. 1-4 can serve as “subject sequence”, while the amino acid sequenceof a bacterial lipocalin different from Blc as described herein servesas “query sequence”.

Given the above, a skilled artisan is thus readily in a position todetermine which amino acid position mutated in Blc as described hereincorresponds to an amino acid of a bacterial lipocalin scaffold otherthan Blc. Specifically, a skilled artisan can align the amino acidsequence of a mutein as described herein, in particular a Blc mutein ofthe invention with the amino acid sequence of a different bacteriallipocalin to determine which amino acid(s) of said mutein correspond(s)to the respective amino acid(s) of the amino acid sequence of saiddifferent lipocalin.

When used herein “a bacterial lipocalin other than Blc” or “a bacteriallipocalin different from Blc” in which one or more amino acidreplacements, in particular at one or more position in one, two, threeor all four loops, can be made in accordance with the teaching of thepresent invention encompasses any other bacterial lipocalin known in theart or which can be identified by using Blc as reference sequence, forexample, in a BLAST search or using a nucleic acid molecule encoding Blcas probe in, for example, a hybridization experiment.

Preferred bacterial lipocalin scaffolds other than Blc in which one ormore amino acid replacements, in particular at one or more position inone, two, three or all four loops, can be made in accordance with theteaching of the present invention can, for example, be retrieved fromthe bacteria shown in the following Table.

A bacterial lipocalin scaffold other than Blc can be retrieved via theaccession number from the bacteria listed in the following Table asfollows: A TBLASTN search (Altschul et al., cited herein) using the Blcprotein sequence is to be performed on both the microbial genomedatabases and the non-redundant database at the NCBI web site(http://www.ncbi.nlm.nih.gov). The genome sequence 500 bp upstream anddownstream of each hit should be retrieved. The six resultingtranslations are evaluated to identify both the full-length lipocalinsequence and the presence of the 16s ribosomal RNA binding site.

Accession Bacterial species Number Agrobacterium tumefaciens NC_003063Caulobacter crescentus NC_002696 Mesorhizobium loti NC_002678Rhodobacter sphaeroides AAAE01000158 Rhodospirillum rubrum AAAG0200000Silicibacter NZ_AAFG01000010 Acidovorax (partial) AB044565 BordetellaNC_002927 Bordetella parapertussis NC_002928 Burkholderia cepaciaAAEH01000003 Burkholderia fungorum NZ_AAAJ03000001 Chromobacteriumviolaceum NC_005085 Dechloromonas aromatica AADF01000001 Methylobacillusflagellatus AADX01000001 Ralstonia eutropha AADY01000001 Rubrivivaxgelatinosus AAEM01000005 Thiobacillus denitrificans AAFH01000001Citrobacter braakii (partial) AF492447 Citrobacter freundii U21727Citrobacter murliniae AJ607409 Enterobacter nimpressuralis AJ487975Erwinia carotovora BX950851 Escherichia coli P39281 Francisellatularensis AY774926 Idiomarina loihiensis NC_006512 Klebsiella oxytocaY17716 Pseudomonas AABQ0700000 Pseudomonas AAAT0300000 Pseudomonasputida NC_002947 Pseudomonas syringae AABP02000002 Salmonella typhiNC_006511 Salmonella typhimurium AE008903 Shewanella oneidensis AE015615Shigella flexneri NC_004741 Vibrio cholerae NC_002506 Rubrivivaxgelatinosus AAEM0100000 Thiobacillus denitrificans AAFH01000001Acinetobacter CR543861 Azotobacter vinelandii NZ_AAAU02000004Citrobacter braakii (partial) AF492447 Citrobacter freundii U21727Citrobacter murliniae AJ607409 Enterobacter AJ487975 Erwinia carotovoraBX950851 Escherichia coli P39281 Francisella tularensis AY774926Idiomarina loihiensis NC_006512 Klebsiella oxytoca Y17716 Pseudomonasaeruginosa AABQ07000004 Pseudomonas fluorescens AAAT03000001 Pseudomonasputida NC_002947 Acinetobacter CR543861 Azotobacter vinelandiiNZ_AAAU02000004 Vibrio parahaemolyticus NC_004605 Vibrio vulnificusNC_004460 Xanthomonas axonopodis (1) NC_003919 Xanthomonas campestris(1) NC_003902 Bdellovibrio NC_005363 Desulfotalea psychrophila NC_006138Geobacter sulfurreducens NC_002939 Campylobacter jejuni AL139078Campylobacter lari NZ_AAFK01000002 Chlorobium tepidum NC_002932Bacteroides fragilis NC_006347 Bacteroides NC_004663 Cytophagahutchinsonii AABD03000002 Parachlamydia NC_005861 Gloeobacter violaceusNC_005125 Corynebacterium efficiens NC_004369 Corynebacterium glutamicumNC_003450 Nocardia farcinica NC_006361 Bacillus subtilis P54945

The present invention also relates to a nucleic acid coding for the oneor more mutein (s) of the bacterial lipocalin protein, which nucleicacid results from mutagenesis. Preferably, said nucleic acid is operablyfused at the 3′end with a gene coding for the coat protein pIII of afilamentous bacteriophage of the M13-family or coding for a fragment ofthis coat protein, in order to select at least one mutein for thebinding of the given target.

In another aspect, the present invention relates a bacterial lipocalinmutein as described herein which is conjugated to a label selected fromthe group consisting of an organic molecule, an enzyme label,radioactive label, fluorescent label, chromogenic label, luminescentlabel, a hapten, digoxigenin, biotin, metal complexes, metals, andcolloidal gold.

In yet another aspect, the present invention relates to a fusion proteincomprising a bacterial lipocalin mutein of the present invention,wherein an enzyme, a protein or a protein domain, a peptide, a signalsequence and/or an affinity tag is operably fused to the amino terminusor the carboxy terminus of said bacterial lipocalin mutein.

Also, the present invention relates to a nucleic acid molecule encodingsaid fusion protein.

Moreover, the present invention relates to a pharmaceutical compositioncomprising a bacterial lipocalin mutein as described herein or a fusionprotein as described herein and a pharmaceutically acceptable carrier orexcipient.

Furthermore, the present invention relates to a method for producing abacterial lipocalin mutein or a fusion protein thereof, wherein themutein or the fusion protein thereof is produced starting from thenucleic acid encoding the mutein by means of genetic engineering methodsin a bacterial or eukaryotic host organism and is isolated from thishost organism or its culture.

Finally, the present invention contemplates a use of a mutein ofbacterial lipocalin or a fusion protein thereof for the detection of agiven target, comprising the steps of contacting the mutein with asample suspected of containing the given target under suitableconditions, thereby allowing formation of a complex between the muteinand the given target, and determining the complexed mutein by a suitablesignal. The given target may be a protein or protein domain, a peptide,a nucleic acid molecule, an organic molecule or a metal complex and thedetection is preferably carried out for validation of the protein aspharmacological drug target.

All references cited herein are hereby incorporated in their entirety.

The present invention is further illustrated by, though in no waylimited to, the following examples.

EXAMPLES Example 1 Recombinant Expression and X-Ray StructureDetermination of Blc

Blc was secreted as a soluble protein into the periplasm of E. coliafter its original type II signal peptide had been exchanged by the typeI signal peptide of OmpA (Ghrayeb et al., 1984), which has proven usefulfor recombinant protein export. In addition, the Cys residue at position1 of the mature polypeptide, which otherwise carries the lipid anchor ofthe natural lipoprotein, was replaced by Ala and the unpaired internalthiol residue Cys 113 was substituted by Ser. Purification from thebacterial periplasmic extract was achieved via streptavidin affinitychromatography employing the Strep-tag II (Schmidt & Skerra, 2007),which had been appended to the C-terminus. Blc was finally obtained as ahomogeneous protein by preparative gel filtration.

During this purification step we noted that our recombinant Blc elutesas a fully monomeric protein. This was confirmed by analytical sizeexclusion chromatography (SEC; data not shown), revealing an apparentsize of 13.0 kDa, which was even smaller than the calculated mass of19.1 kDa for the mature protein and clearly indicating the absence of adimer. Consequently, its oligomerization behaviour was furtherinvestigated by means of analytical ultracentrifugation (AUC), resultingin a monomeric molecular mass of 18.7±0.4 kDa. This demonstrated thatour recombinant Blc forms a stable monomer in solution, at least up to aconcentration of about 40 μM.

Crystallization of Blc was achieved at pH 7.5 with PEG 10000 asprecipitant. The obtained crystals belonged to the space group I4₁22containing one molecule per asymmetric unit. These crystals showed alattice packing different from the previously described crystals ofBlc-X in space group P2₁2₁2₁ with two molecules in the asymmetric unit.Interpretable main chain electron density for Blc was observed for 149of 168 residues present in the construct. Missing residues comprised theN-terminal amino acids 1 to 8 and the C-terminal amino acids 158 to 168,i.e. the entire Strep-tag II.

The overall structure of the recombinant Blc analyzed here was verysimilar to the one of Blc-X (PDB entries 1QWD and 2ACO), revealing thetypical lipocalin fold characterized by a β-barrel with eightanti-parallel strands (designated A-H) and a C-terminal α-helix (FIG.1). In contrast, however, our structure clearly lacked the first twoartificial β-stands (designated −1 and 1, respectively) outside theβ-barrel that were previously described for Blc-X. This Blc variant,Blc-X, carried 18 additional residues at the N-terminus (thereof 9visible in the crystallographic model; PDB entry 2ACO) as well as 4amino acid replacements at the beginning of the mature sequence, bothoriginating from the attB1 Gateway® recombination sequence on theexpression vector pDest17 (Campanacci et al., 2006). Together with astretch of the following native sequence, a two-stranded extraantiparallel β-sheet is formed in the crystal structure of Blc-X (FIGS.1 and 2). The recombinant Blc prepared in the present study, however,lacked this extra peptide segment and had an almost native N-terminus,except for the missing lipid anchor at Cys 1 (data not shown).

Superposition of Blc with the two non-symmetrical monomers A and B ofthe Blc-X crystal structure (PDB entry 2ACO) resulted in an r.m.s.d.(over 149 Cα positions) of 0.68 Å and 0.89 Å, respectively, while mutualsuperposition of the latter two monomers yielded an r.m.s.d. of 1.07 Å.Beside the artificial N-terminus in Blc-X, the largest conformationaldifferences were observed in the E/F-loop, which was well ordered in ourstructure. This loop, which connects strands E and F of the β-barrel atits open end, adopted a distinct conformation in each of the three X-raystructures, apparently influenced by the differing crystal-packingenvironment (FIG. 1). Further deviations were observed at the loopregions connecting β-strands A-B and C-D, indicating increasedflexibility, which has also been described for other members of thisprotein family, for example human tear lipocalin (Breustedt et al.,2009). The differing conformations of the A/B- and E/F-loops arecritical for ligand binding as their arrangement restricts theaccessibility of the deep ligand pocket. Only in monomer B of the Blc-Xcrystal structure the A/B- and E/F-loops adopt a conformation thatallows ligand binding. In our Blc structure and in monomer A of theBlc-X structure, the cavity is mostly shielded by the three Phe residues35, 90, and 91.

Example 2 Reassessment of the Proposed Dimerization Mechanism for Blc

Previous studies of Blc-X indicated a dimeric state both in the crystallattice as well as in solution, suggesting formation of an asymmetricfunctional homodimer with different affinities of its two subunits forlipid ligands (Campanacci et al., 2004; Campanacci et al., 2006). Incontrast, we observed Blc as a monomeric protein not only in the newcrystal form, but also in solution. To thoroughly compare the previouslypublished Blc-X dimer interface with similar crystal packing contacts inour Blc structure, we performed molecular surface and interactionanalyses using PISA (Krissinel & Henrick, 2007).

The dimer interface of Blc-X (PDB entry 2ACO) is formed by 19 and 22residues for molecules A and B, respectively, involving the N-terminalextra peptide segment together with the E/F loop and leading to 709 Å²and 792 Å² buried surface area (BSA). These numbers are smaller than theones of 786 Å² and 825 Å² reported before (Campanacci et al., 2006),which may be attributed to different, in part undisclosed, algorithmsused. However, an even larger discrepancy was observed for the totalsolvent accessible surface area (ASA) of the two Blc-X monomers. UsingPISA, we calculated a total ASA of 8635 and 8784 Å², for molecules A andB, respectively, compared with the published number of 7800 Å² permonomer (Campanacci et al., 2006). Similar larger values were obtainedwith the programs DSSP (Kabsch & Sander, 1983) and AreaIMol (CCP4,1994).

Based on our calculations, on average 8.6% of the total ASA of Blc-Xbecomes buried at the interface of the two monomers A and B. Incontrast, in the new I4₁22 crystals Blc forms its tightest contact witha symmetry-related monomer in a neighbouring unit via a differentsurface region, around the N-terminus of strand A and the preceding loopthat crosses the bottom of the β-barrel (not shown). This contact isaccompanied by a significantly smaller BSA of 569 Å², which correspondsto merely 7.0% of the total ASA (8077 Å²).

A striking feature of the previously described Blc-X dimer interface isthe two-stranded antiparallel β-sheet that originates from theartificial 22 N-terminal residues. This small extra β-sheet itselfneither interacted with the second monomer nor was involved ininteractions with symmetry-related neighbours.

However, the hairpin loop that connects the two β-strands seemed to beimportant for dimer formation of Blc-X (FIG. 2): the loop of molecule Bintimately interacted with molecule A, while the same loop of molecule Adid not participate in an equivalent interaction, in line with theasymmetry of the dimer noted before (Campanacci et al., 2006). When allthe artificially introduced N-terminal residues of molecule B wereomitted from the surface analysis, the BSA became significantly reducedto 608 and 686 Å² for molecules A and B, respectively, that is 7.0% and8.0% of the total ASA. These smaller values would hardly be significantfor a true oligomeric state (Miller et al., 1987). Moreover, the latternumbers were in a similar range as another crystal contact in theP2₁2₁2₁ space group with BSAs of 618 Å² and 606 Å² for molecules A andB, respectively, corresponding to 7.2% and 7.0% of ASA.

Taken together, two structural features became evident for Blc-X: (i)its N-terminal extension gives rise to the additional β-sheet, whichconformationally fixes the loop in between, and (ii) substitution of thefirst four residues Cys-Ser-Ser-Pro of wild-type Blc by Lys-Ala-Gly-Serwithin this loop leads to a unique intermolecular interaction (FIG. 2)which possibly also stabilizes Blc-X dimer formation in solution. Incontrast, the C-terminal Strep-tag II, employed for affinitypurification of Blc in our study, is far away from the N-terminus (atleast 35 Å distance) and structurally disordered, thus clearly lackingdefined interactions with neighbouring molecules in the I4₁22 crystallattice.

The dimer interface of Blc-X was further dominated by the interactionbetween the E/F-loops of monomers A and B, which contributed 375 and 422Å² BSA, respectively, corresponding to ca. 53% of the total contactregion including the N-terminal hairpin loop. Due to the asymmetry ofthe dimer the E/F loop adopted a distinct conformation in each monomer(FIG. 1). Analysis of the alternative crystal packing of Blc in thespace group I4₁22 revealed that a comparable but distinct contactoccurred there with a symmetry mate, related via a crystallographictwo-fold axis (data not shown). This crystal contact showed a total BSAof 480 Å² and was again dominated by the E/F-loop (residues 88-96), witha local BSA of 336 Å² on each molecule corresponding to 70% of the totalBSA in this region. Notably, in the new Blc structure the E/F-loopshowed a conformation different from both Blc-X monomers (FIG. 1),suggesting structural flexibility. Therefore, dimerization via theE/F-loop should be entropically disfavoured in solution. The fact that acrystal contact involving the E/F-loop was observed in both crystalstructures may be solely attributed to its largely hydrophobic nature.

Example 3 Comparison of Blc to ApoD

Mammalian apolipoprotein D (ApoD) is the closest eukaryotic homologue ofBlc and also anchored in a lipid micelle, albeit via a differentmechanism (Eichinger et al., 2007).

Structural comparison between Blc and ApoD resulted in 139 matchingCα-positions out of 149 resolved residues in the Blc structure, with anoverall r.m.s.d. of 1.37 Å (data not shown). Differences between the twolipocalin structures were mainly observed in the strand connecting loopregions at the open end of the β-barrel. The loops A/B and, inparticular, G/H showed the largest deviations. Loop G/H formed anextended hairpin structure in ApoD resulting in a wider pocket, ideallyshaped for accommodation of a steroid ligand. On the other hand, the A/Bloop, which has a one-residue insertion in Blc compared to ApoD,partially shielded the ligand pocket in the bacterial counterpart.Beside variations in pocket size and accessibility, there weredifferences in surface hydrophobicity. While ApoD showed distincthydrophobic patches, which were likely involved in high densitylipoprotein (HDL) micelle association (Eichinger et al., 2007),hydrophobic surface areas of Blc were mainly confined to the interior ofits cavity. This is in agreement with Blc's presumed function to bindfatty acid-like ligands (Campanacci et al., 2006) whereas membraneassociation by its N-terminal lipid anchor.

Example 4 Blc Vector Construction

The coding sequence for Blc was amplified from genomic DNA of E. coliK12 strain TG1/F⁻ (Kim et al., 2009) via PCR according to a publishedprocedure (Skerra, 1992) by using phosphorothioate primers 5′-CCG CCAGTT CTC CTA CGC CGC CG-3′ (also introducing the Cys1 to Ala mutation)(SEQ ID NO. 5) and 5′-GCT ACC AGG CTG CTG TAC CC-3′ (SEQ ID NO. 6). Theunique amplification product was purified by agarose gelelectrophoresis, phosphorylated with T4 polynucleotide kinase (NewEngland Biolabs, Beverly, Mass.), and ligated with the expression vectorpASK75-strepII (Skerra, 1994; Schmidt & Skerra, 2007), which had beencut with StuI and Eco471II and dephosphorylated using shrimp alkalinephosphatase (USB, Cleveland, Ohio). After transformation of E. coliXL1-Blue (Bullock et al., 1987) the resulting plasmid, designated pBlc1,was isolated and its composition was confirmed by restriction digest aswell as double-stranded dideoxy-sequencing (ABI PRISM 310 GeneticAnalyzer; Applied Biosystems, Foster City, Calif.). On pBlc1, therecombinant protein was encoded in fusion with the amino-terminal type Isignal peptide of OmpA and the C-terminal Strep-tag II of nine residues(Breustedt et al., 2006; Schmidt & Skerra, 2007). The codon for theunpaired internal thiol residue Cys113 (numbering according to themature full length protein; Swiss-Prot entry P0A901) was subsequentlyreplaced by a Ser codon via site-directed mutagenesis (Geisselsoder etal., 1987) with the oligodeoxynucleotide 5′-GGT CCG GCC CGC TAA CCA GCGCAT G-3′ (SEQ ID NO. 7), finally yielding pBlc2, which was used forrecombinant protein production throughout this study.

Example 5 Blc Protein Production and Purification

Recombinant Blc was produced in the E. coli K-12 strain JM83(Yanisch-Perron et al., 1985) harbouring pBlc2 by secretion as a solubleprotein into the bacterial periplasm. Shake flask cultures were grown in21 LB medium supplemented with 100 mg/l ampicillin at 22° C. Geneexpression was induced at a cell density of OD₅₅₀=0.5 by adding 0.2 mg/lanhydrotetracycline (Skerra, 1994). After further shaking for 3 h thecells were harvested by centrifugation, resuspended in 500 mM sucrose, 1mM EDTA, 100 mM Tris-HCl pH 8.0, and kept on ice for 30 min. Theresulting spheroplasts were sedimented by centrifugation and thesupernatant containing the recombinant protein was recovered. Theprotein extract was dialyzed against 150 mM NaCl, 1 mM EDTA, 100 mMTris-HCl pH 8.0 and applied to a Strep-Tactin affinity column (Schmidt &Skerra, 2007) using the same buffer. The recombinant Blc wascompetitively eluted by application of 2.5 mM D-desthiobiotin in thechromatography buffer. Elution fractions were concentrated, applied to apreparative Superdex 75 gel filtration column (GE Healthcare, Uppsala,Sweden) using 150 mM NaCl, 1 mM EDTA, 100 mM Tris-HCl pH 8.0 as runningbuffer, and eluted in a homogeneous peak. The yield was ca. 1.5 mg ofpurified protein per 1 l E. coli culture.

Example 6 Blc Biochemical Characterization

Analytical size exclusion chromatography (SEC) was carried out on aTricorn S75 column (Superdex 75 10/300 GL, bed volume V_(t)=24 ml; GEHealthcare) at a flow rate of 0.5 ml/min using ÄKTA Purifierinstrumentation (GE Healthcare) with PBS (4 mM KH₂PO₄, 16 mM Na₂HPO₄,115 mM NaCl) as running buffer. Bovine serum albumin (66 kDa,V_(r)=9.861 ml), carbonic anhydrase (29 kDa, V_(r)=12.35 ml), myoglobin(17.05 kDa, V_(r)=13.244 ml), cytochrome C (12.4 kDa, V_(r)=14.11 ml),and aprotinin (6.5 kDa, V_(r)=16.35 ml) were used as protein sizestandards for calibration of the column while the void volume wasdetermined with blue dextran (V₀=8.08 ml).

Sedimentation equilibrium experiments were performed using an XL-Ianalytical ultracentrifuge and a Ti-60 rotor equipped with a UV/Vis aswell as interference detector (Beckman, Fullerton, Calif.). An 0.8 mg/mlsolution of the purified recombinant Blc in 150 mM NaCl, 1 mM EDTA, 100M Tris/HCl pH 8.0 was applied to six-sector 12 mm path length cells. Thesamples were centrifuged at 25000 rpm for 72 h at 4° C., untilequilibrium was reached, whereby the protein gradient was measured by UVabsorption at 280 nm. Data analysis was carried out with Kaleidagraphsoftware (Synergy Software, Reading, Pa.) as previously described(Zander et al., 2007; Stromer et al., 2004) using a value of 0.73 ml/gfor the specific volume of the protein.

Example 7 Blc Crystallization and Structure Determination

Blc crystals were grown in hanging drops using the vapour diffusiontechnique. Drops mixed from 1 μl protein solution (10 mg/ml, dialyzedagainst 10 mM Tris-HCl pH 8.0) and 1 μl reservoir solution wereequilibrated against 0.5 ml reservoir solution on siliconized glasscover slips. After about two months at 20° C., two crystals wereobtained in the presence of 20% (w/v) PEG 10000, 100 mM HEPES-NaOH pH7.5. Blc crystals were harvested using Nylon loops (Hampton Research,Laguna Niguel, Calif.), cryo-protected with Paratone N (HamptonResearch)—thereby removing excess mother liquor—and frozen in a 100 Knitrogen stream (Oxford Cryosystems, Oxford, UK).

A native data set was collected on a mar345 imaging plate detector(MarResearch, Hamburg, Germany) using monochromatic Cu—K_(α) radiationfrom a RU-300 rotating anode generator (Rigaku, Tokyo, Japan) equippedwith Confocal Max-Flux Optics (Osmic, Troy, Mich.). Diffraction datawere processed with the XDS Package (Kabsch, 1993). The Blc crystalsbelonged to the space group I4₁22 with unit cell parameters a=b=88.94 Å,and c=78.35 Å, containing one protein molecule per asymmetric unit(Table 1). The X-ray structure was solved by molecular replacement asimplemented in PHASER (Storoni et al., 2004) using the coordinates of apublished Blc structure (PDB code 1QWD) after deleting the N-terminalresidues −17 to 4 as well as loop residues 33 to 38 and 60 to 69 at theopen end of the o-w barrel. Model building was performed with Coot(Emsley & Cowtan, 2004), followed by restrained and TLS refinement usingREFMAC5.5 (Murshudov et al., 1997; Winn et al., 2001). Total B valueswere calculated with TLSANL (Howlin et al., 1993). Finally, thestructure was validated with Coot and MolProbity (Davis et al., 2007).

Graphics were prepared with PyMOL (DeLano, 2002) while secondarystructure elements were assigned with DSSP (Kabsch & Sander, 1983).Superposition of structures was performed with SUPERPOSE (Krissinel &Henrick, 2004) and interfaces were analyzed with PISA (Krissinel &Henrick, 2007). The coordinates and structure factors for the refinedBlc structure have been deposited at the RCSB Protein Data Bank (PDBaccession code 3MBT).

REFERENCES

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TABLE 1 Data collection and refinement statistics for Blc. Datacollection Space group l4₁22 Unit cell parameters [Å] a = b = 88.94, c =78.35 Wavelength [Å] 1.5418 Resolution [Å] 30-2.6 (2.7-2.6)^(a)Completeness [%] 98.7 (99.8) Unique reflections 5024 (531) Multiplicity6.1 (6.2) Mean l/σ(l) 34.7 (9.4) R_(meas) [%]^(b) 4.5 (20.1) WilsonB-factor [Å²] 36.4 Refinement Resolution [Å] 18.70-2.60 (2.67-2.60)^(a)Reflections (working) 4787 (343) Reflections (test) 236 (16) R_(cryst)[%]^(c) 22.2 (30.6) R_(free) [%]^(d) 27.6 (46.0) Number of proteinatom/water molecules 1209/18 B-values of protein atoms/water mols. [Å²]36.0/11.7 Ramachandran plot: favoured/outliers [%] 93.2/0.0 Rmsd bonds[Å]/angles [°] 0.009/1.227 ^(a)Values in parentheses represent thehighest resolution shell.$\;^{b}R_{meas} = \frac{\sum\limits_{hkl}\;\sqrt{\frac{n}{{n - 1}\;}{\sum\limits_{j = 1}^{n}\;{{I_{{hkl},j} - \left\langle I_{hkl} \right\rangle}}}}}{\sum_{hkl}{\sum_{j}I_{{hkl},j}}}$$\;^{c}R_{cryst} = \frac{\sum\limits_{hkl}\;{{F_{hkl}^{obs} - F_{hkl}^{calc}}}}{\sum\limits_{hkl}F_{hkl}^{obs}}$^(d)R_(free) is calculated as for R_(cryst), but with 5% of thereflections excluded from the refinement

What is claimed is:
 1. An isolated crystalline form of a monomericBacterial Lipocalin (Blc) polypeptide comprising amino acids 22-189 ofSEQ ID NO:1, wherein said crystalline has a I4122 space group and unitcell dimensions of a=b=88.9 Angstroms and c=78.4 Angstroms.